Polymer linkers and their uses

ABSTRACT

Provided herein are poly-1-hydroxymethylethylene hydroxymethyl formal (PHF)-based drug delivery systems. Also disclosed are methods of making antibody-drug conjugates and methods of treatment using these conjugates.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application, pursuant to 35 U.S.C. §121, of U.S. patent application Ser. No. 15/572,720, filed Nov. 8, 2017,which is the U.S. national phase application, pursuant to 35 U.S.C. §371, of PCT International Application Ser. No. PCT/US2017/035698, filedJun. 2, 2017, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Ser. No. 62/345,557 filed Jun. 3, 2016,the entire contents of which are hereby incorporated by referenceherein.

BACKGROUND

Antibody-drug conjugates are a class of therapeutics that connect anantibody to a drug via a linker. The antibody serves as a drug deliverysystem to a cell expressing an antigen recognized by the antibody.Linkers such as poly-1-hydroxymethylethylene hydroxymethyl formal(“PHF”) have been used in this type of drug delivery system. Highlyhydrophilic polyacetal-based PHF polymers can be utilized as a linker toattach multiple hydrophobic drugs to the antibody without affecting thephysicochemical properties of the antibody or drug. However, existingPHF-based linkers have significant limitations.

In known PHF-based linkers, drugs or small molecules are attached to thePHF backbone through the acylation of hydroxyl groups on the PHFresulting in ester linkages at the acylation sites. Such PHF-based drugsor small molecules are disclosed in U.S. Pat. No. 8,685,383, herebyincorporated by reference in its entirety. However, these newly formedester linkage can undergo enzymatic cleavage upon administration to asubject. Additionally, these ester linkages are also cleaved under basicconditions. Moreover, existing PHF-based technology utilizes a secondcleavable ester linkage, in addition to the ester linkage of hydroxylgroups on PHF resulting in two enzymatically cleavable sitescomplicating various mechanistic processes for the compounds. Multipleenzymatically cleavable sites on the linker of antibody-drug conjugatesmay decrease antitumor activity and increase the risk of toxicity due topremature and nontargeted release of the drug from the antibody. Suchpremature release may narrow the therapeutic window. Moreover, multiplecleavable sites generally make pharmacokinetics studies more challengingdue to more complex kinetic action occurring in linked compounds.Accordingly, delivery of drug payloads with ester linkers can thus beunreliable and difficult to reproduce.

Thus, there is a need in the art to identify non-cleavable linkers thatserve as effective antibody-drug conjugates to deliver drugs in areliable and reproducible manner. The presently disclosed linkers andmethods meet this need.

SUMMARY

The drawbacks of PHF-based linkers can be overcome by using anon-cleavable linkage between the drug and the PHF polymer as describedherein. When a cleavable linker is needed, an optimizable and cleavablemoiety can be introduced in the linkage as required. However, thesynthesis of the presently disclosed compounds allows for independentcontrol of the number and type of cleavable linkers between thetargeting moiety and the polymer backbone. Additionally, the control ofthe number and type of cleavable linkers between the therapeutic agentand the polymer backbone is also possible. Such control mitigatesseveral complications associated with known PHF antibody drugconjugates.

PHF has a high solubility in water, but very limited solubility innon-polar organic solvents. PHF is a polyol, so selective chemicaltransformations of hydroxyl groups on PHF and purifications arechallenging. Moreover, PHF contains pH-sensitive acetal groups, and isdegradable in acidic conditions. Therefore, selective, mild and cleanchemistry is typically required for introducing a non-cleavable linkageto PHF. Also provided herein are methods for synthesizing PHF compoundscapable of producing the antibody-drug conjugates. In some embodiments,the synthesis of the compounds described herein comprises:

-   -   (a) reacting PHF with an electrophilic reagent to form a polymer        comprising an activating group capable of displacement by a        thiol;    -   (b) displacing said activating group by a thiol comprising a        linkage capable of covalently bonding (or undergoing a reaction        to covalently bond) to a drug or a small molecule.        In some embodiments, the synthesis comprises the synthetic steps        shown in Example 1. In some embodiments, the synthesis further        comprises the step of converting a portion of the monomer units        comprising said linkage into a second linkage capable of        covalently bonding (or undergoing a reaction to covalently bond)        to a targeting moiety. In some embodiments, the synthesis        comprises the synthetic steps shown in Example 6 or Example 7.

The PHF conjugates described herein, which may be produced by any of thedescribed synthetic methods comprise block repeat block monomer units(a) and/or (b) and/or (c) and/or (d)

L₁ is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof;L₂ is absent, or can be of the formula:

L_(2A) is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, —C(O)—,—N(R_(C))—, and any combination thereof;L_(2B) and L_(2C) are independently absent or a linker group selectedfrom alkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene,heteroarylene, amidoalkylene, amidoheteroalkylene, —C(O)—, —N(R_(C))—,and any combination thereof;B_(2A) and B_(2B) are independently absent or a cleavable linker;T is a therapeutic agent selected from the group consisting ofchemotherapeutic agents, microtubule inhibitors, DNA-damaging agents andRNA transcription inhibitors;L₃ is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof;R_(e) is a substituent selected from hydrogen, alkyl and heteroalkyl;L₄ is a group of the formula:

L_(4A) is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, —C(O)—,—N(R_(C))—, and any combination thereof;L_(4B) and L_(4C) are independently absent or a linker group selectedfrom alkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene,heteroarylene, amidoalkylene, amidoheteroalkylene, —C(O)—, —N(R_(C))—,and any combination thereof;B_(4A) and B_(4B) are independently absent or a cleavable linker;C_(4A) is a group selected from

whereA is —H or a targeting moiety selected from the group consisting of anantibody, a synthetically functionalized antibody, a peptide and atargeting ligand;“n” is independently at each occurrence an integer ranging from 0-5;R_(c) and R_(d) are independently selected at each occurrence fromhydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl;where each monomer is substituted independently from any additionalmonomer;with the proviso that the compound of Formula (I) contains one or moretherapeutic agents, T, and one or more targeting moieties A. In someembodiments, the PHF compounds comprise blocks of polymerized monomers(a), (b), (d) and optionally (c):

wherein “a” is independently at each occurrence an integer from 1 toabout 3000 (e.g., about 1-about 2000, etc.);“b” is independently at each occurrence an integer from 1 to about 500;“c” is absent or independently at each occurrence an integer from 1 toabout 500;“d” is independently at each occurrence an integer from 1 to about 200;andeach block of monomer unit (a), (b), (c), and (d), is covalentlyattached to at least one block monomer unit (a), (b), (c), and/or (d)and each block of monomer units is independently substituted from anyother block of monomer units. Exemplary cleavable linkers (e.g.,biodegradable linkers, etc.) for B_(2A), B_(2B), B_(4A) and B_(4B) mayinclude —S—S—, —C(═O)O—, —OC(═O)—, —C(═O)NR—, —N(R_(c))C(═O)—,—OC(═O)O—, —NR_(c)C(═O)O—, —OC(═O)N(R_(c))— or —N(R_(c))C(═O)N(R_(d))—,—C(═O)N(R_(c))C(═O)—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—,—SC(═O)O—, —OC(═S)O—, —SC(═S)S—, —N(R_(c))SO₂—, —SO₂N(R_(c))—,—N(R_(c))SO₂N(R_(d))—, —C(═O)N(R_(c))N(R_(d))—, —N(R_(c))N(R_(d))C(═O)—,—N(R_(c))N(R_(d))C(═O)O—, —OC(═O)N(R_(c))N(R_(d))—,—C(R_(c))═N—NH—C(═O)—, —C(═O)NH—N═C(R_(c))—, —C(R_(c))═N—O—,—O—N═C(R_(c))—,

Typically, L_(2A), L_(2B), L_(2C), and combinations thereof (i.e., whenB_(2A) and/or B_(2B) are absent) are non-biodegradable linker moieties.Similarly, L_(4A), L_(4B), and L_(4C) and combinations thereof (i.e.,when L_(4A) and/or L_(4B) are absent) may be non-biodegradable linkermoieties. In some embodiments, L₂ does not comprise a cleavable linker.

The compounds may also have the repeat block monomer units (a), (b), and(e)

L₁ is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof;L₂ is absent, or can be of the formula:

L_(2A) is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, and anycombination thereof;L_(2B) and L_(2C) are independently absent or a linker group selectedfrom alkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene,heteroarylene, amidoalkylene, amidoheteroalkylene, and any combinationthereof;B_(2A) and B_(2B) are independently absent or a cleavable linker;T is a therapeutic agent selected from the group consisting ofchemotherapeutic agents, microtubule inhibitors, DNA-damaging agents andRNA transcription inhibitors;L₃ is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof; L₄ is a group of theformula:

L_(4A) is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, and anycombination thereof;L_(4B) and L_(4C) are independently absent or a linker group selectedfrom selected from alkylene, heteroalkylene, cycloalkylene,heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene and any combination thereof;B_(4A) and B_(4B) are independently absent or a cleavable linkerC_(4A) is a group selected from

A is —H or a targeting moiety selected from the group consisting of anantibody, a synthetically functionalized antibody, a peptide and atargeting ligand;“n” is independently at each occurrence an integer ranging from 0-5;R_(c) and R_(d) are independently selected at each occurrence fromhydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl;where each monomer is substituted independently from any additionalmonomer;with the proviso that the compound of Formula (II) contains one or moretherapeutic agent and one or more targeting moiety. In some embodiments,the compound may comprise blocks of polymerized monomers (a), (b), and(e):

wherein “a” is independently at each occurrence an integer from 1-1860;“b” is independently at each occurrence an integer from 1-372;“e” is independently at each occurrence an integer from 1-186; andeach block of monomer unit (a), (b), and (e), is covalently attached toat least one block monomer unit (a), (b), and/or (e); andeach block of monomer units is independently substituted from any otherblock of monomer units. Typically, L_(2A), L_(2B), L_(2C), andcombinations thereof (i.e., when B_(2A) and/or B_(2B) are absent) arenon-biodegradable linker moieties. Similarly, L_(4A), L_(4B), and L_(4C)and combinations thereof (i.e., when L_(4A) and/or L_(4B) are absent)may be non-biodegradable linker moieties. In some embodiments, L₂ doesnot comprise a cleavable linker.

Pharmaceutical compositions are also described comprising any of thecompounds or pharmaceutically acceptable salts or solvates thereof. Thepharmaceutical compositions may be used in a method of inhibiting cancercells comprising contacting the cancer cells with a pharmaceuticalcomposition comprising an effective amount of one or more compounds. Thepharmaceutical composition may also be used in a method for treating orinhibiting cancer in a patient comprising contacting cancer cells withan anti-cancer effective amount of a pharmaceutical compositioncomprising the compounds. In some embodiments, the treatment maycomprise administering an effective amount of the pharmaceuticalcomposition to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of exemplary embodiments can bebetter understood when read in conjunction with the appended drawings.It should be understood, however, that the disclosure is not limited tothe precise arrangements and instrumentalities of the embodiments shownin the drawings.

FIG. 1 depicts a dose response curve of the % surviving HCC 1954 cellstreated with Auristatin F, Compounds 14-17 and Cisplatin.

FIG. 2 depicts a dose response curve of the % surviving NCI-N87 cellstreated with Auristatin F, Compounds 14-17 and Cisplatin.

FIG. 3 depicts a dose response curve of the % surviving SKBR3 cellstreated with Auristatin F, Compounds 14-17 and Cisplatin.

FIG. 4 depicts a dose response curve of the % surviving BT-474 cellstreated with Auristatin F, Compounds 14-17 and Cisplatin.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure pertains. Thefollowing references provide one of skill with a general definition ofmany of the terms used in this disclosure: Singleton et al., Dictionaryof Microbiology and Molecular Biology (2nd ed. 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary ofGenetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); andHale & Marham, The Harper Collins Dictionary of Biology (1991).Generally, the nomenclature used herein and the laboratory procedures inmedicine, organic chemistry and polymer chemistry are those well-knownand commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, such as ±5%, such as±1%, and such as ±0.1% from the specified value, as such variations areappropriate to perform the disclosed methods.

As used herein, the term “alkyl”, by itself or as part of anothersubstituent means, unless otherwise stated, a branched or unbranchedsaturated hydrocarbon group. The term “n-alkyl” refers to an unbranchedalkyl group. The term “C_(x)-C_(y) alkyl” refers to an alkyl grouphaving between x and y carbon atoms, inclusively, in the branched orunbranched hydrocarbon group. By way of illustration, but withoutlimitation, the term “C₁-C₈ alkyl” refers to a straight chain orbranched hydrocarbon moiety having from 1, 2, 3, 4, 5, 6, 7, or 8 carbonatoms. “C₁-C₆” refers to a straight chain or branched hydrocarbon moietyhaving from 1, 2, 3, 4, 5, or 6 carbon atoms. “C₁-C₄ alkyl” refers to astraight chain or branched hydrocarbon moiety having from 1, 2, 3, or 4carbon atoms, including methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and tert-butyl. The term “C₁-C₄ n-alkyl” refers tostraight chain hydrocarbon moieties that have 1, 2, 3, or 4 carbon atomsincluding methyl, ethyl, n-propyl, and n-butyl. An “alkylene” is analkyl substituent which is covalently bound to two substituents or asingle substituent twice.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings)wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples includephenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, mostpreferred is phenyl. An “arylene” is an aryl substituent which iscovalently bound to two substituents or a single substituent twice.

As used herein, the term “heteroalkyl”, by itself or as part of anothersubstituent means, unless otherwise stated, a branched or unbranchedalkyl group in which one or more carbon atoms in the main chain havebeen substituted with heteroatoms. The heteroatoms include, but notlimited to, oxygen, sulfur, silicon, phosphorus, nitrogen atoms, or acombination thereof. A “heteroalkylene” is a heteroalkyl substituentwhich is covalently bound to two substituents or a single substituenttwice.

As used herein, the term “amidoalkyl” refers to an alkyl group that hasa —C(O)NR_(a)— or —NR_(a)C(O)— group at either terminus of the alkylgroup or within the alkyl group. For example, R_(a) is selected from H,alkyl and heteroalkyl. An “amidoalkylene” is an amidoalkyl substituentwhich is covalently bound to two substituents or a single substituenttwice.

As used herein, the term “amidoheteroalkyl” refers to an heteroalkylgroup that has a —C(O)NR_(a)— or —NR_(a)C(O)— group at either terminusof the heteroalkyl group or within the chain of the heteroalkyl group.For example, R_(a) is selected from H, alkyl and heteroalkyl. An“amidoheteroalkylene” is an amidoheteroalkyl substituent which iscovalently bound to two substituents or a single substituent twice.

As used herein, the term “alkoxy” by itself or as part of anothersubstituent means, unless otherwise stated, an —O-alkyl group, includingfrom 1 to 10 carbon atoms of a straight, branched, saturated cyclicconfiguration and combinations thereof, attached to the parent molecularstructure through an oxygen. Examples include methoxy, ethoxy, propoxy,isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy, cyclohexyloxy andthe like. In some embodiments, an alkoxy group can have one to sixcarbons denoted C₁-C₃. In some embodiments, C₁₋₄ alkoxy is an alkoxygroup which encompasses both straight and branched chain alkyls of from1 to 4 carbon atoms. In some aspects, the alkoxy group is a(C₁-C₃)alkoxy, such as, but not limited to, ethoxy and methoxy.

As used herein, the term “heterocycle” or “heterocyclyl” or“heterocyclic” by itself or as part of another substituent means, unlessotherwise stated, an unsubstituted or substituted, stable, mono- ormulti-cyclic heterocyclic ring system that consists of carbon atoms andat least one heteroatom selected from the group consisting of N, O, andS, and wherein the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen atom may be optionally quarternized. Theheterocyclic system may be attached, unless otherwise stated, at anyheteroatom or carbon atom that affords a stable structure. A heterocyclemay be aromatic or non-aromatic in nature. In one embodiment, theheterocycle is a heteroaryl. A “heterocyclylene” is a heterocyclylsubstituent which is covalently bound to two substituents or a singlesubstituent twice.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includetetrahydroquinoline and 2,3-dihydrobenzofuryl. A “heteroarylene” is aheteroaryl substituent which is covalently bound to two substituents ora single substituent twice.

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl(particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl,pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl,pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles include indolyl (particularly 3-,4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl,isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

The aforementioned listing of heterocyclyl and heteroaryl moieties isintended to be representative and not limiting.

As used herein, the term “8” with reference to nuclear magneticresonance results refers to the measured chemical shifts for measurednucleii. Unless otherwise specified, 6 is in units of ppm.

As used herein, the term “DMSO” refers to dimethylsulfoxide.

As used herein, the term “halo” or “halogen” employed alone or as partof another substituent means, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom, such as fluorine, chlorine, orbromine, further such as, fluorine or chlorine.

As used herein, “hydroxyl” refers to —OH.

As used herein, the term “reaction condition” refers to a physicaltreatment, chemical reagent, or combination thereof, which is requiredor optionally required to promote a reaction. Non-limiting examples ofreaction conditions are electromagnetic radiation, heat, a catalyst, achemical reagent (such as, but not limited to, an acid, base,electrophile or nucleophile), and a buffer.

As used herein, the term “salt” refers to a salt of a compoundcontemplated herein, including inorganic acids, organic acids, inorganicbases, organic bases, solvates, hydrates, or clathrates thereof. As usedherein, the term “salt” embraces addition salts of free acids or freebases that are compounds useful in the methods described herein. In somecases, undesired salts may nonetheless possess properties such as highcrystallinity, which may have utility in the practice of the methodsdescribed herein, such as, for example, utility in process of synthesisor purification of compounds described herein.

Suitable acid addition salts may be prepared from an inorganic acid oran organic acid. Examples of inorganic acids include hydrochloric,hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids,perchloric and tetrafluoroboronic acids. Appropriate organic acids maybe selected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxy-ethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid. Suitable base additionsalts of disclosed compounds include, for example, metallic saltsincluding alkali metal, alkaline earth metal and transition metal saltssuch as, for example, lithium, calcium, magnesium, potassium, ammonium,sodium and zinc salts. Acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-dibenzyl-ethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All ofthese salts may be prepared by conventional means from the correspondingfree base compound by reacting, for example, the appropriate acid orbase with the corresponding free base.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. Unless stated otherwise, any group recited herein may besubstituted.

For aryl and heterocyclyl groups, the term “substituted” as applied tothe rings of these groups refers to any level of substitution, namelymono-, di-, tri-, tetra-, or penta-substitution, where such substitutionis permitted. The substituents are independently selected, andsubstitution may be at any chemically accessible position. In oneembodiment, the substituents vary in number between one and four. Inanother embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two. In yet another embodiment, the substituents areindependently selected from the group consisting of C₁-C₆ alkyl, —OH,C₁-C₆ alkoxy, halo, amino, acetamido and nitro. As used herein, where asubstituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic, such as straight.

As used herein, the term “targeting moiety” is a chemical moiety able tobind to a biological entity. The term targeting moiety may refer to achemical species such as an antibody, an enzyme, a protein or peptide orany other biological binding ligand.

The term “effective amount” or “therapeutically effective amount” of anagent, as used herein, is that amount sufficient to effect beneficial ordesired results, such as clinical results, and, as such, an “effectiveamount” depends upon the context in which it is being applied. Forexample, in the context of administering an agent that is an anticanceragent, an effective amount of an agent is, for example, an amountsufficient to achieve alleviation or amelioration or prevention orprophylaxis of one or more symptoms or conditions; diminishment of theextent of cancer, disorder, or condition; stabilized (i.e., notworsening) state of cancer, disorder, or condition; preventing spread ofcancer, disorder, or condition; delay or slowing the progress of thedisease, disorder, or condition; amelioration or palliation of thedisease, disorder, or condition; and remission (whether partial ortotal), whether detectable or undetectable, as compared to the responseobtained without administration of the agent.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein formulated with apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is manufactured or sold with the approval ofa governmental regulatory agency as part of a therapeutic regimen forthe treatment of disease in a mammal. Pharmaceutical compositions can beformulated, for example, for oral administration in unit dosage form(e.g., a tablet, capsule, caplet, gelcap, or syrup); for topicaladministration (e.g., as a cream, gel, lotion, or ointment); forintravenous administration (e.g., as a sterile solution free ofparticulate emboli and in a solvent system suitable for intravenoususe); or in any other formulation described herein (see below).

Useful pharmaceutical carriers for the preparation of the compositionshereof, can be solids, liquids, or gases. Thus, the compositions cantake the form of tablets, pills, capsules, suppositories, powders,enterically coated or other protected formulations (e.g., binding onion-exchange resins or packaging in lipid-protein vesicles), sustainedrelease formulations, solutions, suspensions, elixirs, and aerosols. Thecarrier can be selected from the various oils including those ofpetroleum, animal, vegetable or synthetic origin, e.g., peanut oil,soybean oil, mineral oil, and sesame oil. Water, saline, aqueousdextrose, and glycols are preferred liquid carriers, particularly (whenisotonic with the blood) for injectable solutions. For example,formulations for intravenous administration comprise sterile aqueoussolutions of the active ingredient(s) which are prepared by dissolvingsolid active ingredient(s) in water to produce an aqueous solution, andrendering the solution sterile. Suitable pharmaceutical excipientsinclude starch, cellulose, talc, glucose, lactose, talc, gelatin, malt,rice, flour, chalk, silica, magnesium stearate, sodium stearate,glycerol monostearate, sodium chloride, dried skim milk, glycerol,propylene glycol, water, and ethanol. The compositions may be subjectedto conventional pharmaceutical additives such as preservatives,stabilizing agents, wetting or emulsifying agents, salts for adjustingosmotic pressure, and buffers. Suitable pharmaceutical carriers andtheir formulation are described in Remington's Pharmaceutical Sciencesby E. W. Martin. Such compositions will, in any event, contain aneffective amount of the active compound together with a suitable carrierso as to prepare the proper dosage form for administration to therecipient.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose.

It will be understood that the description of compounds herein islimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding with regard to valencies, etc., andto give compounds which are not inherently unstable. For example, anycarbon atom will be bonded to two, three, or four other atoms,consistent with the four valence electrons of carbon.

Throughout this disclosure, various aspects of the disclosure may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thepresent claims. Accordingly, the description of a range should beconsidered to have specifically disclosed all the possible sub-ranges aswell as individual numerical values within that range and, whenappropriate, partial integers of the numerical values within ranges. Forexample, description of a range such as from 1 to 6 should be consideredto have specifically disclosed sub-ranges such as from 1 to 3, from 1 to4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and so forth, aswell as individual numbers within that range, for example, 1, 2, 2.7, 3,4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Compounds and Compositions

Provided herein are drug delivery systems that include an antibody-drugconjugate formed from a polymer backbone, a first linker connected to anantibody and a second linker connected to a therapeutic agent or smallmolecule. In one embodiment, the polymer is apoly-1-hydroxymethylethylene hydroxymethyl formal (PHF)-based polymer.In some embodiments, the first linker includes a sulfide (—S—) bonded tothe polymer which is covalently attached to a targeting moiety, such asan antibody. In some embodiments, the second linker includes a sulfide(—S—) bonded to the polymer which is attached to a therapeutic agent. Inother embodiments, the second linker is attached to a protein. Theprotein may comprise cysteine and/or lysine. In some embodiments,cysteine and/or lysine may be the point of conjugation to the protein.

In certain embodiments, the polymer of the invention is a polyacetal,e.g., a poly-1-hydroxymethylethylene hydroxymethyl formal (PHF). Inother embodiments, the polyacetal has a molecular weight ranging fromabout 10 kDa to 250 kDa.

In certain embodiments, the targeting moieties are selected from thegroup consisting of antibodies, synthetically functionalized antibodies,peptides and other targeting ligands. Examples of targeting antibodiescan include, but are not limited to, monoclonal antibodies that arespecific for antigens which are overexpressed in cancer cells, antigensregulated from driver oncogenes, antigens in tumor stroma andvasculature or antigens found in haematological malignancies. In certainembodiments, the targeting moieties can include, but are not limited to,HER-2, EGFR, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a,mesothelin, ENPP3, guanylyl cyclase C, SLC44A4, NaPi2b, CD70, mucin 1,STEAP1, nectin 4, 5T4, SLTRK6, SC-16, LIV-1, P-Cadherin, PSMA,Fibronectin Extra-domain B, Endothelin receptor ETB, Tenascin c,Collagen IV, VEGFR2, Periostin, CD30, CD79b, CD19, CD22, CD138, CD37,CD33, CD74, CD19 and CD98. In certain embodiments, the targeting moietyis selected from trastuzumab and pertuzumab.

Without being limited to any one theory, the targeting moiety can allowthe therapeutic agents or small molecules to be localized at aparticular targeting site, for example, a tumor or a tissue. This caneffectively increase the efficacy of therapeutic agents at the targetingsite, while minimizing unwanted side-effects on normal cells.

In certain embodiments, the therapeutic agent is a chemotherapeuticagent (e.g., a quinolone alkaloid such as camptothecin, etc.). In someembodiments, the therapeutic agent (e.g., chemotherapeutic agent) caninclude, but is not limited to, microtubule inhibitors, DNA-damagingagents, and RNA transcription inhibitors, and any combinations thereof.In yet other embodiments, the microtubule inhibitors can be one or moreselected from, but not limited to, the group consisting of, auristatin,maytansinoid, taxol derivative, vinca alkaloid and any derivativesthereof. In yet other embodiments, the DNA-damaging agents can be one ormore selected from, but not limited to, the group consisting of acalicheamicin, a duocarmycin, a doxorubicin, a CC-1065 analog, amethotrexate, a pyrrolobenzodiazepine (PBD) and any derivatives thereof.In yet other embodiments, the RNA transcription inhibitor can be anamanitin, including α-amanitin, β-amanitin, γ-amanatin and ε-amanatinand any derivatives thereof. The therapeutic agent may be tublysin,kinase inhibitor, MEK inhibitor, KSP inhibitor or any combinationthereof.

The invention provides an antibody-drug conjugate of Formula (I) asshown below, comprising hydroxy monomeric blocks (a) and/or (b) and/or(c) and/or (d).

“*” indicates a covalent bond to an additional section of free hydroxymonomer of a formula selected from the group consisting of (a), (b), (c)and (d) wherein each monomer is substituted independently from anyadditional monomer;

L₁ is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof;

L₂ is absent, or can be of the formula:

wherein:

L_(2A) is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, and anycombination thereof;

B_(2A) is absent or a cleavable linker selected from —S—S—, —C(═O)O—,—OC(═O)—, —C(═O)NR_(c)—, —NR_(c)C(═O)—, —OC(═O)O—, —NR_(c)C(═O)O—,—OC(═O)NR_(c)— or —NR_(c)C(═O)NR_(d)—, —C(═O)NR_(c)C(═O)—, —C(═O)S—,—SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —OC(═S)O—, —SC(═S)S—,—NR_(c)SO₂—, —SO₂NR_(c)—, —NR_(c)SO₂NR_(d)—, —C(═O)NR_(c)NR_(d)—,—NR_(c)NR_(d)C(═O)—, —NR_(c)NR_(d)C(═O)O—, —OC(═O)NR_(c)NR_(d)—,—CR_(c)═N—NH—C(═O)—, —C(═O)NH—N═CR_(c)—, —CR_(c)═N—O—, and —O—N═CR_(c)—,wherein R_(c) and R_(d) are each a substituent independently selectedfrom hydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl;

L_(2B) can be absent or a linker group selected from alkylene,heteroalkylene, cycloalkylene, heterocyclylene, arylene, heteroarylene,amidoalkylene, amidoheteroalkylene, and any combination thereof;

B_(2B) can be absent or a cleavable linker selected from —S—S—,—C(═O)O—, —OC(═O)—, —C(═O)NR_(c)—, —NR_(c)C(═O)—, —OC(═O)O—,—NR_(c)C(═O)O—, —OC(═O)NR_(c)— or —NR_(c)C(═O)NR_(d)—,—C(═O)NR_(c)C(═O)—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—,—OC(═S)O—, —SC(═S)S—, —NR_(c)SO₂—, —SO₂NR_(c)—, —NR_(c)SO₂NR_(d)—,—C(═O)NR_(c)NR_(d)—, —NR_(c)NR_(d)C(═O)—, —NR_(c)NR_(d)C(═O)O—,—OC(═O)NR_(c)NR_(d)—, —CR_(c)═N—NH—C(═O)—, —C(═O)NH—N═CR_(c)—,—CR_(c)═N—O—, and —O—N═CR_(c)—, wherein R_(c) and R_(d) are each asubstituent independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, and heterocyclyl;

L_(2c) is absent or a linker group selected from alkylene,heteroalkylene, cycloalkylene, heterocyclylene, arylene, heteroarylene,amidoalkylene, amidoheteroalkylene, and any combination thereof;

T is a therapeutic agent;

L₃ is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof;

R_(e) is a substituent selected from hydrogen, alkyl and heteroalkyl;

L₄ is a group of the formula:

wherein:

L_(4A) is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, and anycombination thereof;

B_(4A) is absent or is a cleavable linker selected from —S—S—, —C(═O)O—,—OC(═O)—, —C(═O)NR_(c)—, —NR_(c)C(═O)—, —OC(═O)O—, —NR_(c)C(═O)O—,—OC(═O)NR_(c)— or —NR_(c)C(═O)NR_(d)—, —C(═O)NR_(c)C(═O)—, —C(═O)S—,—SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —OC(═S)O—, —SC(═S)S—,—NR_(c)SO₂—, —SO₂NR_(c)—, —NR_(c)SO₂NR_(d)—, —C(═O)NR_(c)NR_(d)—,—NR_(c)NR_(d)C(═O)—, —NR_(c)NR_(d)C(═O)O—, —OC(═O)NR_(c)NR_(d)—,—CR_(c)═N—NH—C(═O)—, —C(═O)NH—N═CR_(c)—, —CR_(c)═N—O—, —O—N═CR_(c)—,wherein R_(c) and R_(d) are each a substituent independently selectedfrom hydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl;

L_(4B) is absent or is a linker group selected from alkylene,heteroalkylene, cycloalkylene, heterocyclylene, arylene, heteroarylene,amidoalkylene, amidoheteroalkylene and any combination thereof,

B_(4B) is absent or is a cleavable linker selected from —S—S—, —C(═O)O—,—OC(═O)—, —C(═O)NR_(c)—, —NR_(c)C(═O)—, —OC(═O)O—, —NR_(c)C(═O)O—,—OC(═O)NR_(c)— or —NR_(c)C(═O)NR_(d)—, —C(═O)NR_(c)C(═O)—, —C(═O)S—,—SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —OC(═S)O—, —SC(═S)S—,—NR_(c)SO₂—, —SO₂NR_(c)—, —NR_(c)SO₂NR_(d)—, —C(═O)NR_(c)NR_(d)—,—NR_(c)NR_(d)C(═O)—, —NR_(c)NR_(d)C(═O)O—, —OC(═O)NR_(c)NR_(d)—,—CR_(c)═N—NH—C(═O)—, —C(═O)NH—N═CR_(c)—, —CR_(c)═N—O—, —O—N═CR_(c)—,wherein R_(c) and R_(d) are each a substituent independently selectedfrom hydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl;

L_(4c) is absent or is a linker group selected from alkylene,heteroalkylene, cycloalkylene, heterocyclylene, arylene, heteroarylene,amidoalkylene, amidoheteroalkylene and any combination thereof;

C_(4A) is a linker group selected from:

A is a targeting moiety, as described above, or H;

n is an integer ranging from 0-5

a is an integer from 1-1860;

b is an integer from 1-372;

c is an integer from 0-465;

d is an integer from 1-186; and

with the proviso that the antibody-drug conjugate of Formula (I) mustcontain one or more therapeutic agent and one or more targeting moiety.

The invention also provides an antibody-drug conjugate of Formula (II)as shown below, comprising hydroxy monomeric blocks (a), (b) and (e):

wherein *, L₁, L₂, T, L₃, L₄ and A are as described above;

a is an integer from 1-1860;

b is an integer from 1-372;

e is an integer from 1-186;

with the proviso that the antibody-drug conjugate of Formula (II) mustcontain one or more therapeutic agent and one or more targeting moiety.

In certain embodiments, cleavable linkers B_(2A), B_(2B), B_(4A) andB_(4B) can be enzymatically cleaved, be biodegradable, or can be cleavedby changes in pH (e.g. acid or base labile). Linkers that are cleavableunder reducing or oxidizing conditions may also be used (review: Jain etal., Pharm. Res. 2015, 32, Pages 3526-3540). Cleavable linkers may beselected from, but are not limited to, one or more of the followingstructures:

In other embodiments, the linker L₂ does not have a cleavable linkage(B_(2A) and B_(2B) are both absent) and the therapeutic agent, T, can becleaved off by antibody degradation (See U.S. Publication No.2005/0238649 and reference herein). In some embodiments, monomer unit(b) has the structure:

In certain embodiments, the targeting moiety possesses a nucleophilicgroup, allowing it to react with the electrophilic C_(4A). In otherembodiments, the targeting moiety comprises a site-specific modifiednon-natural amino acid with a chemical side chain that allows forbiorthogonal conjugation chemistry with C_(4A). In certain embodiments,the targeting moiety is modified with an azide or alkyne to allow for[3+2] cycloaddition with an alkyne or azide, respectively, on C_(4A).

In yet other embodiments, the targeting moiety (A) can be linked toL_(4C) through a cross-linking reagent selected from the groupconsisting of N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC),maleimide-polyethyleneglycol-N-hydroxysuccinimide,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyliodoacetate (SLA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl3-(bromoacetamido)propionate (SBAP).

Typically, the mole ratio of the therapeutic agent (T) to targetingmoiety (A) is generally greater than 1:1. In some embodiments, the ratiois greater than about 5:1 or greater than about 8:1 or greater thanabout 10:1 or greater than about 12:1 or greater than about 15:1 orgreater than about 18:1. In some embodiments, the ratio is between about1:1 to about 20:1 (e.g., between about 5:1 to about 15:1, between about8:1 to about 13:1, etc.).

In some embodiments, the compound of Formula (I) is Compound 16,Compound 17, Compound 25, or Compound 30:

The invention also provides auristatin derivatives of Formula (III):

wherein L_(T) is a linking moiety selected from —(CH₂)_(m)—,—(OCH₂)_(m)—, —(OCH₂CH₂)_(m)—, and —(CH₂CH₂O)_(m)—, “m” is an integerfrom 0 (i.e. L_(T) is a bond) to 6; andR_(f) is selected from hydrogen, —NH₂, —C(O)—NH₂,—[C(R_(c))(R_(d))]_(p)—NH₂, —C(O)—[C(R_(c))(R_(d))]_(p)—NH₂,

and “p” is an integer from 1-4. In some embodiments, the auristarinderivative has the structure:

wherein “m” is an integer from 1-6; andR_(f) is selected from:

In some embodiments the auristarin derivative may be connected to a PHFpolymer via a contacting the R_(f) moiety with a polymer with a suitablefunctional group to form a covalent bond between the polymer and theauristarin derivative. In some embodiments, R_(f) comprises the point ofattachment of said camptothecin derivative to said compound. R_(f) maybe hydrogen (and if R_(f) is hydrogen and the point of attachment, thenit is a bond) or

The invention also provides camptothecin derivatives having thestructure of formula (IV):

wherein L_(T) is a linking moiety selected from —(CH₂)_(m)—,—(OCH₂)_(m)—, —(OCH₂CH₂)_(m)—, and —(CH₂CH₂O)_(m)—, “m” is an integerfrom 0 (i.e. L_(T) is a bond) to 6; andR_(f) is selected from hydrogen, —NH₂, —C(O)—NH₂,—[C(R_(c))(R_(d))]_(p)—NH₂, —C(O)—[C(R_(c))(R_(d))]_(p)—NH₂,

and “p” is an integer from 1-4. In some embodiments the camptothecinderivative may be connected to a PHF polymer via a contacting the R_(f)moiety with a polymer with a suitable functional group to form acovalent bond between the polymer and the camptothecin derivative. Insome embodiments, R_(f) comprises the point of attachment of saidcamptothecin derivative to said compound. R_(f) may be hydrogen (and ifR_(f) is hydrogen and the point of attachment, then it is a bond) or

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the (R_(c)) or(S) configuration. In certain embodiments, compounds described hereinare present in optically active or racemic forms. The compoundsdescribed herein encompass racemic, optically active, regioisomeric andstereoisomeric forms, or combinations thereof that possess thetherapeutically useful properties described herein. Preparation ofoptically active forms is achieved in any suitable manner, including byway of non-limiting example, by resolution of the racemic form withrecrystallization techniques, synthesis from optically active startingmaterials, chiral synthesis, or chromatographic separation using achiral stationary phase. A compound illustrated herein by the racemicformula further represents either of the two enantiomers or mixturesthereof, or in the case where two or more chiral center are present, alldiastereomers or mixtures thereof.

In certain embodiments, the compounds of the invention exist astautomers. All tautomers are included within the scope of the compoundsrecited herein.

Compounds described herein also include isotopically labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, substitution withheavier isotopes such as deuterium affords greater chemical stability.Isotopically labeled compounds are prepared by any suitable method or byprocesses using an appropriate isotopically labeled reagent in place ofthe non-labeled reagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

In all of the embodiments provided herein, examples of suitable optionalsubstituents are not intended to limit the scope of the claimedinvention. The compounds of the invention may contain any of thesubstituents, or combinations of substituents, provided herein.

Salts

The compounds described herein may form salts with acids or bases, andsuch salts are included in the present invention. The term “salts”embraces addition salts of free acids or bases that are useful withinthe methods of the invention. The term “pharmaceutically acceptablesalt” refers to salts that possess toxicity profiles within a range thataffords utility in pharmaceutical applications. In certain embodiments,the salts are pharmaceutically acceptable salts. Pharmaceuticallyunacceptable salts may nonetheless possess properties such as highcrystallinity, which have utility in the practice of the presentinvention, such as for example utility in process of synthesis,purification or formulation of compounds useful within the methods ofthe invention.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic,hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (includinghydrogen phosphate and dihydrogen phosphate). Appropriate organic acidsmay be selected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,sulfanilic, 2-hydroxyethanesulfonic, trifluoromethanesulfonic,p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic,β-hydroxybutyric, salicylic, galactaric, galacturonic acid,glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate).Salts may be comprised of a fraction of one, one or more than one molarequivalent of acid or base with respect to any compound of theinvention.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, ammonium salts and metallic saltsincluding alkali metal, alkaline earth metal and transition metal saltssuch as, for example, calcium, magnesium, potassium, sodium and zincsalts. Pharmaceutically acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (or N-methylglucamine) and procaine. All ofthese salts may be prepared from the corresponding compound by reacting,for example, the appropriate acid or base with the compound.

Methods

The invention also provides a method of inhibiting cancer cells, themethod comprising administering an anti-cancer effective amount of acomposition comprising a compound of Formula (I). In another embodiment,provided herein is a method of inhibiting cancer cells, the methodcomprising administering an anti-cancer effective amount of acomposition comprising a compound of Formula (II). In certainembodiments, the invention provides a method of treating or inhibitingcancer in a subject, the method comprising administering a compositioncomprising a compound of Formula (I) or Formula (II) to a subject inneed thereof.

In some embodiments, the cancer can be HER2-positive cancer. In someembodiments, the cancer can be breast cancer. In some embodiments, thecancer can be HER2-positive breast cancer.

In some embodiments of the method, the HER2 is overexpressed in breastcancer cells. In some instances, the compound of Formula (I) or Formula(II) comprises an anti-neopalastic agent, such as Auristatin F, linkedto the PHF polymer, such as in compound 16 or 17. In some embodiments,the compound of Formula (I) or Formula (II) includes an antibody, suchas Trastuzumab, linked to the PHF polymer. Trastuzumab is known to bindto the extracellular domain of HER2 protein in HER2 overexpressingbreast cancer cells. The compounds of the invention can enable the drugto be targeted to the cancer cell, thus minimizing off-target activityor toxicity.

In certain embodiments, the methods of the invention compriseadministering a compound of Formula (I) or Formula (II) as part of apharmaceutical composition, further comprising one or more additionalingredients. In some embodiments the compounds of the invention can bepackaged as a lyophilized cake which can be reconstituted with a volumeof sterile water.

In certain embodiments, the methods of the invention compriseadministering a compound of Formula (I) or Formula (II) in combinationwith another compound or therapeutic agent such as an anti-tumor agent,chemotherapeutic, anti-cell proliferation agent or any combinationthereof. In certain embodiments, the methods of the invention compriseadministering a compound of Formula (I) or Formula (II) in combinationwith another standard breast cancer treatment method including, but notlimited to, surgical intervention and radiation therapy.

In certain embodiments of the methods, the compounds of the inventioncan be administered to a subject in a dosage of between 1 ng/kg/day and500 mg/kg/day. In some preferred embodiments, the compounds of theinvention can be administered to a subject in a dosage of between 0.1mg/kg to about 10 mg/kg. In some embodiments, the compounds areadministered daily. In some embodiments, the compounds are administeredevery other day. In some embodiments, the compounds are administeredonce a week, once every other week, once a month or once every othermonth. In some embodiments, the compounds are administered as part of astandard chemotherapy regimen.

Synthesis

The compounds can be prepared from commercially available startingmaterials, compounds known in the literature, or readily preparedintermediates, by employing standard synthetic methods and proceduresknown to those skilled in the art. The compounds may also be preparedfrom the synthetic schema outlined in Examples 1-27. Standard syntheticmethods and procedures for the preparation of organic molecules andfunctional group transformations and manipulations can be readilyobtained from the relevant scientific literature or from standardtextbooks in the field. It will be appreciated that where typical orpreferred process conditions (i.e., reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures. Those skilled in the art of organic synthesiswill recognize that the nature and order of the synthetic stepspresented may be varied for the purpose of optimizing the formation ofthe compounds described herein.

Synthetic chemistry transformations (including protecting groupmethodologies) useful in synthesizing the compounds described herein areknown in the art and include, for example, those such as described in R.C. Larock, Comprehensive Organic Transformations, 2d. Ed., Wiley-VCHPublishers (1999); P. G. M. Wuts and T. W. Greene, Protective Groups inOrganic Synthesis, 4th Ed., John Wiley and Sons (2007); L. Fieser and M.Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wileyand Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995), and subsequent editionsthereof.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy (FT-IR),spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or bychromatography such as high performance liquid chromatography (HPLC) orthin layer chromatography (TLC).

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein byreference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includespreparation of the Mosher's ester or amide derivative of thecorresponding alcohol or amine, respectively. The absolute configurationof the ester or amide is then determined by proton and/or ¹⁹F NMRspectroscopy. An example method includes fractional recrystallizationusing a “chiral resolving acid” which is an optically active,salt-forming organic acid. Suitable resolving agents for fractionalrecrystallization methods are, for example, optically active acids, suchas the D and L forms of tartaric acid, diacetyltartaric acid,dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or thevarious optically active camphorsulfonic acids. Resolution of racemicmixtures can also be carried out by elution on a column packed with anoptically active resolving agent (e.g., dinitrobenzoylphenylglycine).Suitable elution solvent compositions can be determined by one skilledin the art.

Pharmaceutical Compositions and Formulations

The invention also encompasses the use of pharmaceutical compositions ofat least one compound of the invention or a salt thereof to practice themethods of the invention.

Such a pharmaceutical composition may consist of at least one compoundof the invention or a salt thereof, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise at least one compound of the invention or a salt thereof, andone or more pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The at least one compound ofthe invention may be present in the pharmaceutical composition in theform of a physiologically acceptable salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

In certain embodiments, the pharmaceutical compositions useful forpracticing the method of the invention may be administered to deliver adose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for inhalational, oral, rectal,vaginal, parenteral, topical, transdermal, pulmonary, intranasal,buccal, ophthalmic, intrathecal, intravenous or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations. Theroute(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of at least one compound ofthe invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it may include isotonic agents, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. A non-limiting preservative is a combination ofabout 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition preferably includes an antioxidant and a chelating agentwhich inhibit the degradation of the compound. Exemplary antioxidantsfor some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid inthe preferred range of about 0.01% to 0.3%, for example BHT in the rangeof 0.03% to 0.1% by weight by total weight of the composition. Thechelating agent may be present in an amount of from 0.01% to 0.5% byweight by total weight of the composition. Exemplary chelating agentsinclude edetate salts (e.g. disodium edetate) and citric acid in theweight range of about 0.01% to 0.20%, for example in the range of 0.02%to 0.10% by weight by total weight of the composition. The chelatingagent is useful for chelating metal ions in the composition which may bedetrimental to the shelf life of the formulation. While BHT and disodiumedetate are the particularly preferred antioxidant and chelating agentrespectively for some compounds, other suitable and equivalentantioxidants and chelating agents may be substituted therefore as wouldbe known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl cellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Administration/Dosing

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after surgical intervention related to cancer.Further, several divided dosages, as well as staggered dosages may beadministered daily or sequentially, or the dose may be continuouslyinfused, or may be a bolus injection. Further, the dosages of thetherapeutic formulations may be proportionally increased or decreased asindicated by the exigencies of the therapeutic or prophylacticsituation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat cancer in the patient. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the activity of the particular compound employed; thetime of administration; the rate of excretion of the compound; theduration of the treatment; other drugs, compounds or materials used incombination with the compound; the state of the disease or disorder,age, sex, weight, condition, general health and prior medical history ofthe patient being treated, and like factors well-known in the medicalarts. Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. A non-limiting example of an effectivedose range for a therapeutic compound of the invention is from about0.01 and 50 mg/kg of body weight/per day. One of ordinary skill in theart would be able to study the relevant factors and make thedetermination regarding the effective amount of the therapeutic compoundwithout undue experimentation.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of cancer in a patient.

In one embodiment, the compositions of the invention are administered tothe patient in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regimen and the precise dosage and composition tobe administered to any patient will be determined by the attendingphysical taking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 mg to about 7,500 mg, about 20 mg to about 7,000 mg, about40 mg to about 6,500 mg, about 80 mg to about 6,000 mg, about 100 mg toabout 5,500 mg, about 200 mg to about 5,000 mg, about 400 mg to about4,000 mg, about 800 mg to about 3,000 mg, about 1 mg to about 2,500 mg,about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mgto about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about150 mg, and any and all whole or partial increments therebetween. Incertain preferred embodiments, the compounds of the invention can beadministered to a subject in a dosage from about 0.1 mg/kg body weightto about 10 mg/kg body weight.

In some embodiments, the dose of a compound of the invention is fromabout 0.5 mg and about 5,000 mg. In some embodiments, a dose of acompound of the invention used in compositions described herein is lessthan about 5,000 mg, or less than about 4,000 mg, or less than about3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 200 mg, or less than about 50 mg. Similarly,in some embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof cancer in a patient.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating, preventing, orreducing cancer in a patient.

Routes of Administration

Routes of administration of any of the compositions of the inventioninclude inhalational, oral, nasal, rectal, parenteral, sublingual,transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal,(trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal,and (trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients which are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmoticallycontrolled release tablets. Tablets may further comprise a sweeteningagent, a flavoring agent, a coloring agent, a preservative, or somecombination of these in order to provide for pharmaceutically elegantand palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propylpara-hydroxy benzoates or sorbic acid). Liquid formulations of apharmaceutical composition of the invention which are suitable for oraladministration may be prepared, packaged, and sold either in liquid formor in the form of a dry product intended for reconstitution with wateror another suitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds usefulwithin the methods of the invention, and a further layer providing forthe immediate release of one or more compounds useful within the methodsof the invention. Using a wax/pH-sensitive polymer mix, a gastricinsoluble composition may be obtained in which the active ingredient isentrapped, ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solutionor as a lyophilized cake which can be reconstituted by the addition of asolvent. This suspension or solution may be formulated according to theknown art, and may comprise, in addition to the active ingredient,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form, in a liposomal preparation, or as acomponent of a biodegradable polymer system. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see U.S. Pat. No.6,323,219).

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another aspect, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active compound should be presentin the amount of from about 0.0001% to about 15% by weight volume of thecomposition. More preferable, it should be present in an amount fromabout 0.0005% to about 5% of the composition; most preferably, it shouldbe present in an amount of from about 0.001% to about 1% of thecomposition. Such compounds may be synthetically- or naturally derived.

Buccal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) of the active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein. Theexamples of formulations described herein are not exhaustive and it isunderstood that the invention includes additional modifications of theseand other formulations not described herein, but which are known tothose of skill in the art.

Rectal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

Controlled Release Formulations and Drug Delivery Systems

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as tablets,capsules, gelcaps, and caplets, which are adapted for controlled-releaseare encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood level of the drug, andthus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “controlled-releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, or microspheresor a combination thereof that facilitates the controlled-release of theactive ingredient.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisdisclosure and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and methods, and are not intended tolimit the scope of what the inventor(s) regard(s) as the invention.

EXAMPLES

The compounds disclosed herein may be synthesized using techniqueswell-known in the art of organic synthesis. The starting materials andintermediates required for the synthesis may be obtained from commercialsources, and/or synthesized according to methods known to those skilledin the art and/or disclosed elsewhere herein.

The following Examples are provided for the purpose of illustrationonly, and the disclosure is not limited to these Examples, but ratherencompasses all variations that are evident as a result of the teachingsprovided herein.

Abbreviations

μl=microliters

Boc or BOC=tert-butoxycarbonyl

DMAP=4-Dimethyl aminopyridine

DMSO=dimethyl sulfoxide

DTT=dithiothreitol

EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ESI or ES=Electrospray ionization

g=grams

h=hour

HPLC=high-performance liquid chromatography

LC=liquid chromatography

LCMS=liquid chromatography mass spectrometry

min=minute

mg=milligrams

ml=milliliters

mmol=millimoles

MS=mass spectrometry

MWCO=molecular weight cut off

NMR=nuclear magnetic resonance spectroscopy

PBS=phosphate-buffered saline, 0.9% NaCl

SPA=3-sulfanylpropionic acid

S SPy=2-(pyridine-2-yldisulfanyl)

TEAA=triethylammonium acetate

TFA=trifluoroacetic acid

Ts or tosyl=p-toluenesulfonyl

Variables *, a, b, c, d and e have the ranges as described above andherein.

Example 1: General Preparation Methods

Poly-1-hydroxymethylethylene hydroxymethyl formal (PHF) 1 may be reactedwith an electrophilic reagent to form polymer 2 where G is an activatinggroup, such as tosyl, methanesulfonyl, or trifluoromethanesulfonyl.Generally, more than 3 must be present in polymer 1. Variables a, b, cand d are as described above. In this described general preparationmethod, z is a+b+c+d which is < or =1862.

The activating group G can be displaced by thiol L-SH to form polymer 3having a sulfide linker. In one embodiment, L is a group that can becovalently linked to a drug or small molecule. In another embodiment, Lis a group already linked to a drug or small molecule.

Alternatively, the sulfide linker can also be formed as follows:

Here, a leaving group X such as bromo or chloro is installed by througha nucleophilic substitution reaction to give polymer 4. A thiol L-SH canthen displace the halide to form the sulfide 3 as shown below.

Example 2: Synthesis of poly(1-carbonylethylene carbonyl formal)(Compound 6)

Dextran (5, 8.0 g, Mn 15 KDa-25 KDa, from Leuconostoc spp.) wasdissolved in 20 ml of deionized water. A solution of sodiummetaperiodate (26.38 g, 0.123 mol) dissolved in 480 ml of deionizedwater was added into the dextran solution at 0-5° C. in a lightprotected flask. The reaction mixture was stirred for 3 h at 0-5° C.,and then at 25° C. for 11 hours. The reaction mixture was desalted usingdiafiltration (Amicon Ultra-15 centrifugal filter, molecular weight cutoff (MWCO): 3K), and concentrated to 60 ml. The pH of the productsolution was adjusted to 8-9 by adding 5.0 N sodium hydroxide solutiondropwise. The poly(1-carbonylethylene carbonyl formal) (compound 6)solution was directly used in the next step.

Example 3: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal) (Compound 1)

Sodium borohydride (4.31 g, 0.113 mol) was added into 10 ml deionizedwater, and stirred for 2 min at 0° C., followed by the addition ofstarting poly(1-carbonylethylene carbonyl formal) (compound 6, 6.42 g,in 60 ml water) at 0° C. The reaction mixture was stirred at 0° C. for 2hours. The pH of the reaction solution was adjusted to pH 7, by slowlyadding 1.0 N aqueous hydrogen chloride solution. The resultant solutionwas desalted by diafiltration, using Amicon Ultra-15 centrifugal filter(MWCO: 3K). The solution was lyophilized to givepoly(1-hydroxymethylethylene hydroxy-methyl formal) (compound 1) as acolorless solid (1.2 g). 1H NMR (400 MHz, DMSO-d₆:D₂O=95:5) δ ppm3.30-3.41 (m, 2H), 3.46 (d, J=4.89 Hz, 2H), 3.60-3.67 (m, 2H), 3.67-3.75(m, 1H), 4.64 (t, J=5.26 Hz, 1H).

Example 4: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-tosyl (Compound 7)

Poly(1-hydroxymethylethylene hydroxy-methyl formal) (compound 1, 0.21 g)was dissolved in anhydrous pyridine (2.2 ml) at 0° C., followed by theaddition of tosyl chloride (90 mg). The reaction mixture was firststirred for one hour 0° C., then warmed to 25° C. and stirred for 16hours. Pyridine was evaporated in vacuo and the product(poly(1-hydroxymethylethylene hydroxy-methyl formal)-tosyl, compound 7)was directly used in the next step, without isolation or purification.

Example 5: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA (Compound 8)

Potassium carbonate (2.14 g, 15.5 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-tosyl (compound 7)in methanol (10.0 ml) at 15° C., followed by the addition of3-mercaptopropionic acid (0.329 g, 0.27 ml, 3.10 mmol). The reactionmixture was stirred at 15° C. for 16 hours. 3-mercaptopropionic acid(0.11 g, 0.09 ml, 1.03 mmol) was added and the reaction mixture washeated to 40° C. for 8 hours. The reaction solution was cooled to roomtemperature and concentrated in vacuo. Water (15.0 ml) was added andstirred for 30 minutes at 15° C. The solid was filtered and the filtratewas desalted by diafiltration, using Amicon Ultra-15 centrifugal filter(MWCO: 3K). The desalted solution was lyophilized to affordpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA (compound 8) asa colorless solid (298 mg). ¹H NMR (400 MHz, D₂O) shows the methylenegroup adjacent to acid (δ ppm 2.39, t, J=6.35 Hz). The3-sulfanylpropionic acid content was found to be 12%, as determined byNMR.

Example 6: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-maleimide (Compound 9)

N-Hydroxysuccinimide (18 mg, 0.155 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA (compound 8, 200mg, 3-sulfanylpropionic acid content: 19%) in deionized water (1 ml) at0° C. EDC (24 mg, 0.027 ml, 0.155 mmol) was added into the reactionsolution at 0° C., followed by the addition of N-(2-aminoethyl)maleimidetrifluoroacetic acid salt (39 mg, 0.155 mmol). The reaction solution waswarmed to 20° C. and stirred for 16 hours. The reaction mixture wasfiltered and the filtrate was desalted by diafiltration, using AmiconUltra-15 centrifugal filter (MWCO: 3K). The desalted solution waslyophilized to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-maleimide (compound 9) as a colorless solid (106 mg). Themaleimide content was found to be 6%, as determined by NMR.

Example 7: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-SSPy (Compound 10)

N-Hydroxysuccinimide (7 mg, 0.062 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA (compound 8, 149mg, 3-sulfanylpropionic acid content: 12%) in deionized water (1 ml) at0° C. EDC (10 mg, 0.011 ml, 0.062 mmol) was added into the reactionsolution at 0° C., followed by the addition of pyridine dithioethylaminehydrochloride (14 mg, 0.062 mmol). The reaction solution was warmed to20° C. and stirred for 16 hours. The reaction mixture was filtered andthe filtrate was desalted by diafiltration, using Amicon Ultra-15centrifugal filter (MWCO: 3K). The desalted solution was lyophilized togive poly(1-hydroxymethyl ethylene hydroxy-methyl formal)-SPA-SSPy(compound 7) as a colorless solid (100 mg). 1H NMR (400 MHz, D₂O) showsthe pyridine group on SSPy: δ ppm 8.33 (br.s, 1H), 7.77 (br.s, 1H), 7.23(br.s, 1H). The SSPy content was found to be 4%, as determined by NMR.

Example 8: Synthesis of auristatin F 2-(2-hydroxy-ethoxy)-ethylamide(Compound 11)

N,N-Diisopropylethylamine (26 mg, 0.034 ml, 0.198 mmol) was added into asolution of auristatin F hydrochloride (52 mg, 0.066 mmol) in anhydrousDMF (1.4 ml) at 10° C., followed by the addition of(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidhexafluorophosphate) (50 mg, 0.132 mmol). Reaction solution was stirredat 10° C. for 15 min, then 2-(2-amino-ethoxy)-ethanol (0.021 mg, 0.020ml, 0.198 mmol) was added and the reaction solution was stirred at 10°C. for 16 hours. The solvent was removed in vacuo, and the residue waspurified by reverse-phase preparatory HPLC to afford auristatin F2-(2-hydroxy-ethoxy)-ethylamide (compound 11, TFA salt, 16.9 mg) as awhite solid. Mass calculated for C₄₄H₇₆N₆O₉+H, [M+H]⁺ 833.57, observedLC/MS (ESI) m/z 833.29 [M+H]⁺, 855.25 [M+Na]⁺.

Example 9: Synthesis of auristatin F 2-(2-hydroxy-ethoxy)-ethylamideBoc-L-Alanine (Compound 12)

Boc-L-Alanine (14 mg, 0.072 mmol) and DMAP (11 mg, 0.09 mmol) wasdissolved in anhydrous dichloromethane (2.0 ml), followed by theaddition of diisopropylcarbodiimide (9 mg, 0.072 mmol) at 0° C.Auristatin F 2-(2-hydroxy-ethoxy)-ethylamide (compound 11, TFA salt,16.9 mg) was added at 0° C., and the reaction mixture was stirred at 23°C. for 21 hours. The reaction mixture was concentrated in vacuo, and theresidue was purified by reverse-phase preparatory HPLC to affordauristatin F 2-(2-hydroxy-ethoxy)-ethylamide Boc-L-Alanine (compound 12,TFA salt, 11.6 mg) as a white solid. Mass calculated for C₅₂H₈₉N₇O₁₂+H,[M+H]⁺ 1004.66, observed LC/MS (ESI) m/z 1004.33 [M+H]⁺, 1026.29[M+Na]⁺.

Example 10: Synthesis of auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine (Compound 13)

Trifluoroacetic acid (0.10 ml) was added dropwise to a solution ofauristatin F 2-(2-hydroxy-ethoxy)-ethylamide Boc-L-Alanine (compound 12,TFA salt, 11.6 mg, 0.01 mmol) in dichloromethane (0.3 ml), and thereaction solution was stirred for one hour at 25° C. The reactionmixture was concentrated in vacuo. The residue was dissolved indichloromethane (1 ml), followed by the addition of ethyl acetate. Theprecipitation was collected to give auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine (compound 12, TFA salt, 10mg). Mass calculated for C₄₇H₈₁N₇O₁₀+H, [M+H]⁺ 904.60, observed LC/MS(ESI) m/z 904.27 [M+H]⁺, 926.30 [M+Na]⁺.

Example 11: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-maleimide (Compound 14)

N-Hydroxysuccinimide (1.5 mg, 0.013 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-maleimide(compound 9, 15 mg, 3-sulfanylpropionic acid content: 13%, maleimidecontent: 6%) in deionized water (0.8 ml) at 10° C. EDC (2.0 mg, 2.3 μl,0.013 mmol) was added into the reaction solution at 10° C., followed bythe addition of auristatin F 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine(compound 13, TFA salt, 5 mg) in acetonitrile (0.4 ml). The reactionsolution was warmed to 23° C. and stirred for 18 hours. The reactionmixture was filtered and concentrated in vacuo. The residue was dilutedwith deionized water (1.0 ml) and desalted by diafiltration, usingAmicon Ultra-15 centrifugal filter (MWCO: 3K). The desalted solution waslyophilized to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-maleimide (compound 14) as a colorless solid. 1H NMR (400MHz, D₂O) shows the phenyl group on auristatin F: δ ppm 7.11-7.31 (m,5H). The auristatin F 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine contentwas found to be 6%, as determined by NMR.

Example 12: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-SSPy (Compound 15)

N-Hydroxysuccinimide (1.5 mg, 0.013 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-SSPy (compound10, 15 mg, 3-sulfanylpropionic acid content: 8%, SSPy content: 4%) indeionized water (0.8 ml) at 10° C. EDC (2.0 mg, 2.3 μl, 0.013 mmol) wasadded into the reaction solution at 10° C., followed by the addition ofauristatin F 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine (compound 13, TFAsalt, 5 mg) in acetonitrile (0.4 ml). The reaction solution was warmedto 23° C. and stirred for 18 hours. The reaction mixture was filteredand concentrated in vacuo. The residue was diluted with deionized water(1.0 ml) and desalted by diafiltration, using Amicon Ultra-15centrifugal filter (MWCO: 3K). The desalted solution was lyophilized togive poly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-(auristatinF 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine)-SSPy (compound 15) as acolorless solid. 1H NMR (400 MHz, D₂O) shows the phenyl group onauristatin F: δ ppm 7.11-7.31 (m, 5H). The auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine content was found to be 4.8%,as determined by NMR.

Example 13: Preparation of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-(Trastuzumab-MCC) (Compound 16)

A solution ofsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) inDMSO (5 μl, 15 mg/ml) was added into a solution of Trastuzumab (5 mg) inTEAA buffer (1.0 ml, pH=7.0). The reaction mixture was stirred for 3hours at 25° C. The reaction mixture was desalted by diafiltration,using Amicon Ultra centrifugal filter (MWCO: 30K) to giveTrastuzumab-MCC. Trastuzumab-MCC was stored in PBS buffer (pH=7.0, 20mg/ml).

Dithiothreitol (DTT, 5.0 mg) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-(auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine)-SSPy (compound 15, 5 mg) indeionized water (0.25 ml). The mixture was stirred at 23° C. for 30 min,the diluted with deionized water (1 ml). The reaction solution waspurified with Amicon Ultra-15 centrifugal filter (cutoff: 3K) to givepoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-(auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine)-SH (stored concentration: 20mg/ml in deionized water).

A solution of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine)-SH(3 mg) in deionized water (150 μl) was added into a solution ofTrastuzumab-MCC (3 mg) in PBS buffer (pH=7.0, 350 μl). The reactionmixture was stirred for 5 hours at 23° C., and purified bysize-exclusion chromatography using Superose-6 column (eluant: PBSbuffer, pH=7.0) to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-(Trastuzumab-MCC) (compound 16). HPLC analysis determinedmolar ratio of auristatin F to Trastuzumab is about 9:1 to 12:1.

Example 14: Preparation of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-Trastuzumab (Compound 17)

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 54 μl, 2.0 mMsolution in TEAA buffer, pH=7.4) was added into a solution ofTrastuzumab (3 mg) in TEAA buffer (400 μl, pH=7.4), and the solution wasincubated at 37° C. for one hour. A solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-(auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine)-maleimide (compound 14, 912μg) in deionized water (45.6 μl) was added, and the solution wasincubated at 25° C. for 6 hours. The product was purified bysize-exclusion chromatography using Superose-6 column (eluant: PBSbuffer, pH=7.0) to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-Trastuzumab (compound 17). HPLC analysis determined molarratio of auristatin F to Trastuzumab is about 12:1 to 15:1.

Example 15: Synthesis of 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(Compound 18)

N-hydroxysuccinimide (105.2 mg, 0.914 mmol) was added into a solution of3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoicacid (300 mg, 0.914 mmol) in 5 ml anhydrous dichloromethane at roomtemperature, followed by the addition of N,N′-dicyclohexylcarbodiimide(198 mg, 0.96 mmol). The reaction mixture was stirred for 2 hours atroom temperature. The white solid formed was filtered and the filtratewas concentrated under vacuum to give the crude product (compound 18,346 mg) which was used directly in the next step.

Example 16: Synthesis of tert-butyl(16-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,14-dioxo-7,10-dioxa-3,13-diazahexadecyl)carbamate(Compound 19)

tert-Butyl (2-aminoethyl)carbamate (139.5 mg, 0.138 ml, 0.871 mmol) wasadded into a solution of 2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate(compound 18) in 4 ml anhydrous acetonitrile at room temperature,followed by the addition of triethyl amine (88.1 mg, 0.121 ml, 0.871mmol). The reaction mixture was stirred at room temperature for 16hours. The mixture was filtered and the filtrate was concentrated undervacuum to give a light yellow oil (compound 19).

Example 17: Synthesis ofN-(2-aminoethyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanamide(Compound 20)

Trifluoroacetic acid (1.0 ml) was added dropwise into a solution oftert-butyl(16-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,14-dioxo-7,10-dioxa-3,13-diazahexadecyl)carbamate(compound 19) in dichloromethane (3.0 ml) and the reaction solution wasstirred for 2 hours at room temperature. The reaction mixture wasconcentrated in vacuo, and the residue was purified by reverse-phasepreparatory HPLC to affordN-(2-aminoethyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanamide(compound 20, TFA salt) as a colorless oil. Mass calculated forC₁₆H₂₆N₄O₆+H, [M+H]⁺ 371.19, observed LC/MS (ESI) m/z 371.43 [M+H]⁺.

Example 18: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-maleimide (Compound 21)

N-Hydroxysuccinimide (2.2 mg, 0.019 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA (compound 8, 30mg, 3-sulfanylpropionic acid content: 10.7%) in deionized water (2 ml)at 20° C.N-(2-aminoethyl)-3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanamide.TFA (9.0 mg, 0.019 mmol) was added into the reaction solution at 20° C.The pH of reaction mixture was adjusted to 6 using 0.05 N NaOH solution.EDC.HCl (4.5 mg, 0.024 mmol) was added and the reaction solution wasstirred 20° C. for 40 min. After 40 min, EDC.HCl (4.5 mg, 0.024 mmol)was added again and the reaction solution was stirred for 18 hours. Thereaction mixture was filtered and the filtrate was desalted bydiafiltration, using Amicon Ultra-15 centrifugal filter (MWCO: 3K). Thedesalted solution was lyophilized to give poly(1-hydroxymethylethylenehydroxy-methyl formal)-SPA-maleimide as a colorless solid (33 mg,compound 21). 1H NMR (400 MHz, D₂O) shows the maleimide group: δ ppm6.76 (s, 2H). The maleimide content was found to be 2.9%, as determinedby NMR.

Example 19: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-maleimide (Compound 22)

N-Hydroxysuccinimide (2.0 mg, 0.014 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-maleimide(compound 21, 16 mg, 3-sulfanylpropionic acid content: 7.8%, maleimidecontent: 2.9%) in deionized water (1.0 ml) at 10° C. Auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine (TFA salt, 14 mg) was addedinto the reaction solution at 10° C. The pH of reaction mixture wasadjusted to 6 using 0.05N NaOH solution. EDC.HCl (4 mg, 0.021 mmol) wasadded and the reaction solution was stirred 20° C. for 40 min. EDC.HCl(4 mg, 0.021 mmol) was added again to the solution and the solution wasthen stirred for 18 hours. The reaction mixture was filtered and thefiltrate was desalted by diafiltration, using Amicon Ultra-15centrifugal filter (MWCO: 3K). The desalted solution was lyophilized togive the product as a white solid (compound 22, 18 mg). 1H NMR (400 MHz,D₂O) shows the phenyl group on auristatin F: δ ppm 7.16-7.26 (m, 5H).The auristatin F 2-(2-hydroxy-ethoxy)-ethylamide L-Alanine content wasfound to be 7%, as determined by NMR.

Example 20: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-maleimide (Compound 23)

N-Hydroxysuccinimide (10 mg, 0.090 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA (compound 8, 86mg, 3-sulfanylpropionic acid content: 21%) in deionized water (3.0 ml)at 20° C. 1-[2-(2-Aminoethoxy)-ethyl]maleimide-HCl (20.0 mg, 0.090 mmol)was added into the reaction solution at 20° C. The pH of reactionmixture was adjusted to 6 using 0.05 N NaOH solution. EDC.HCl (17.5 mg,0.090 mmol) was added and the reaction solution was stirred 20° C. for40 min. EDC.HCl (17.5 mg, 0.090 mmol) was added again to the reactionmixture which was then stirred for 18 hours. The reaction mixture wasfiltered and the filtrate was desalted by diafiltration, using AmiconUltra-15 centrifugal filter (MWCO: 3K). The desalted solution waslyophilized to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-maleimide (compound 23, 89 mg) as a colorless solid. 1H NMR(400 MHz, D₂O) shows the maleimide group: δ ppm 6.76 (s, 2H). Themaleimide content was found to be 5.8%, as determined by NMR.

Example 21: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-maleimide (Compound 24)

N-Hydroxysuccinimide (9.4 mg, 0.066 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-maleimide (75mg, 3-sulfanylpropionic acid content: 15.2%, maleimide content: 5.8%) indeionized water (4.5 ml) at 10° C. Auristatin F2-(2-hydroxy-ethoxy)-ethylamide L-Alanine (TFA salt, 66 mg, 0.065 mmol)was added into the reaction solution at 10° C. The pH of reactionmixture was adjusted to 6 using 0.05 N NaOH solution. EDC.HCl (19 mg,0.099 mmol) was added and the reaction solution was stirred 20° C. for40 min. EDC.HCl (19 mg, 0.099 mmol) was added a second time to thereaction solution which was then stirred for 18 hours. The reactionmixture was filtered and the filtrate was desalted by diafiltration,using Amicon Ultra-15 centrifugal filter (MWCO: 3K). The desaltedsolution was lyophilized to give the product as a white solid (68 mg).1H NMR (400 MHz, D₂O) shows the phenyl group on auristatin F: δ ppm7.18-7.26 (m, 5H). The auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine (compound 24) content was found to be 7.7%, as determined byNMR.

Example 22: Preparation of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-Trastuzumab (Compound 25)

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 510 μl, 1.02 μmol,2.0 mM solution in TEAA, pH=7.4) was added into a solution ofTrastuzumab (30 mg, 0.2895 μmol) in TEAA buffer (1.5 ml, pH=7.4) underAr, and the solution was incubated at 37° C. for two hours. The reactionmixture was cooled to 0° C. The partially reduced Trastuzumab solutionwas added into a solution of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-maleimide (compound 24, 37.5 mg) in deionized water (1.5 ml)at 0° C. The solution was stirred at 0° C. for 30 min, then warmed toroom temperature and stirred for 4 hours. The reaction was quenched withan aqueous solution of cysteine hydrochloride (21 mg). The reactionmixture was stirred at room temperature for one hour. The product waspurified by size-exclusion chromatography using Superose-6 column(eluant: PBS buffer, pH=7.0) to give poly(1-hydroxymethylethylenehydroxy-methyl formal)-SPA-(auristatin F 2-(2-hydroxy-ethoxy)-ethylamideL-Alanine)-Trastuzumab (compound 25). Average ratio of auristatin F toTrastuzumab is about 8.

Example 23: Synthesis of (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (Compound 26)

Di-tert-butyl dicarbonate (144 mg, 0.629 mmol) was added into asuspension of 7-ethyl-10-hydroxy-camptothecin (SN-38, 190 mg, 0.484mmol) in 19 mL of anhydrous dichloromethane, followed by the addition ofanhydrous pyridine (1.157 mL, 14.365 mmol). The reaction suspension wasstirred overnight at room temperature. The suspension was filtered andthe filtrate was extracted with 0.5 N HCl (3×12 mL) and saturated NaHCO₃(1×12 mL). The organic phase was dried over MgSO₄, filtered andevaporated under vacuum to give a pale yellow solid (compound 26, 232mg, yield: 97.3%).

Example 24: Synthesis of tert-butyl(S)-(2-(2-(2-((((9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)ethoxy)ethoxy)ethyl)carbamate(Compound 27)

(S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (compound 29, 0.232 g, 0.471 mmol), DMAP (0.173 g, 1.413mmol), and triphosgene (0.061 g, 0.207 mmol) were added into a roundbottomed flask, followed by the addition of dichloromethane (1.0 mL).The reaction mixture was stirred for a few minutes and monitored by TLC.2-[2-(2-Boc-aminoethoxy)ethoxy]ethanol (0.143 g, 0.575 mmol) was addedinto the above solution. The reaction mixture was stirred for 5 min, andthen purified by flash chromatography using ethyl acetate to givecompound 27 (279 mg, yield: 77%). Mass calculated for C₃₉H₄₉N₃O₁₃+H,[M+H]⁺ 768.3, observed LC/MS (ESI) m/z 768.2 [M+H]⁺, 790.2 [M+Na]⁺.

Example 25: Synthesis of (S)-2-(2-(2-aminoethoxy)ethoxy)ethyl(4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound 28)

tert-Butyl(S)-(2-(2-(2-((((9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)ethoxy)ethoxy)ethyl)carbamate(compound 27, 123 mg, 0.16 mmol) was dissolved in 0.4 mL TFA and stirredfor 5 min at room temperature. To the reaction solution was added 4 mLdiethyl ether and the mixture was stirred for 5 min. The suspension wasfiltered and the solid was collected, and lyophilized to give compound28 (82 mg). Mass calculated for C₂₉H₃₃N₃O₉, [M+H]⁺ 568.2, observed LC/MS(ESI) m/z 568.2 [M+H]⁺, 590.2 [M+Na]⁺.

Example 26: Synthesis of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(7-ethyl-10-hydroxy-camptothecin)-maleimide (Compound 29)

N-Hydroxysuccinimide (4 mg, 0.033 mmol) was added into a solution ofpoly(1-hydroxymethylethylene hydroxy-methyl formal)-SPA-maleimide(compound 8, 50 mg, 3-sulfanylpropionic acid content: 27.6%, maleimidecontent: 3%, 10 K poly(l-hydroxymethylethylene hydroxy-methyl formal))in deionized water (1.0 ml) at 20° C.(S)-2-(2-(2-aminoethoxy)ethoxy)ethyl(4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound 28, TFA salt, 24 mg, 0.036 mmol) was added into thereaction solution at 20° C. The resulting mixture was cooled to 5-10°C., and the pH of reaction mixture was adjusted to 6 using 0.05 N NaOHsolution. EDC.HCl (11 mg, 0.054 mmol) was added and the reactionsolution was stirred at 5-10° C. for 40 min. EDC.HCl (11 mg, 0.054 mmol)was added a second time to the reaction solution and the solution wasstirred for 18 hours at 20° C. The reaction mixture was filtered and thefiltrate was desalted by diafiltration, using Amicon Ultra-15centrifugal filter (MWCO: 3K). The desalted solution was lyophilized togive the product (44 mg). 1H NMR (400 MHz, D₂O) shows the aromatichydrogen on camptothecin: δ ppm 7.41, 6.94. The camptothecin content wasfound to be 8%, as determined by NMR.

Example 27: Preparation of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(7-ethyl-10-hydroxy-camptothecin)-Trastuzumab (Compound 30)

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 120 μl, 0.241 μmol,2.0 mM solution in TEAA, pH=7.4) was added into a solution ofTrastuzumab (10 mg, 0.0687 μmol) in TEAA buffer (0.5 ml, pH=7.4) underAr, and the solution was incubated at 37° C. for two hours. The reactionmixture was cooled to 0° C. The partially reduced Trastuzumab solutionwas added into a solution of poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(7-ethyl-10-hydroxy-camptothecin)-maleimide (compound 29,19.0 mg) in deionized water (1.14 ml) and DMF (50 μl) at 0° C. Thesolution was stirred at 0° C. for 30 min, then warmed to roomtemperature and stirred for 4 hours. The reaction was quenched with anaqueous solution of cysteine hydrochloride (7 mg). The reaction mixturewas stirred at room temperature for one hour. The product was purifiedby size-exclusion chromatography using Superose-6 column (eluant: PBSbuffer, pH=7.0) to give poly(1-hydroxymethylethylene hydroxy-methylformal)-SPA-(7-ethyl-10-hydroxy-camptothecin)-Trastuzumab (compound 30).Average ratio of 7-ethyl-10-hydroxy-camptothecin to Trastuzumab is about16.

Example 28: Cell Viability Assay

Compounds and conjugates were tested for their activity using the CellViability Assay (CellTiter-Glo® Luminescent Cell Viability Assay fromPromega), which measures the number of viable cells in culture aftertreatment with the inventive compounds or conjugates for 72 hours basedon quantitation of the ATP present (Cell Viability. IC₅₀).

Three HER2 expressing breast cancer cell lines (BT474, HCC1954 andSK-BR-3) and HER2 expressing gastric cancer cell line NCI-N87 were usedin the viability assay. The cells were placed in opaque-walled 96-wellplate and allowed to adhere overnight at 37° C. in 5% CO₂ and 95%humidity atmosphere. Cell density per well: 7000 (BT474), 3000(HCC1954), 5000 (NCI-N87), 4000 (SK-BR-3). The test compounds orconjugates were added to experimental wells, and incubate at 37° C. in5% CO₂ and 95% humidity atmosphere for 72 hours. The plates wereequilibrated at room temperature for 30 min. CellTiter-Glo® reagentequal to the volume of cell culture medium present in each well wasadded. After cell lysis on an orbital shaker for 2 min, the plate wasincubated at room temperature for 10 min. Luminescence were recordedusing EnVision® Multilabel Reader (PerkinElmer, 2104-0010A). GraphPadPrism 5.0 was used to analyze the data. The dose response curves weredetermined, and the IC₅₀ value was calculated.

Table 1 provides IC₅₀ data for Compounds 14-17 in the above cell lineswith Auristatin F and Cisplatin as controls. See FIGS. 1-4

TABLE 1 IC₅₀ (nM) Cell Auristatin Cmpd Cmpd Cmpd Cmpd Lines F 15 14 1617 Cisplatin HCC1954 38.2 23.9 32.2 0.13 <0.05 6051.8 NCI-N8769.8 >500 >500 55.6 <0.05 4564.7 SK-BR-3 51 37 48 22 <0.05 1919 BT-474264.1 57.4 87.9 0.39 <0.05 >100000

This data demonstrates that a known antineoplastic agent in HER2 cancercell lines, Auristatin F, is delivered to the cancer cells by theantibody-drug conjugates Compounds 16 and 17. Compounds 14 and 15 lackthe antibody Trastuzumab so the Auristatin F is not targeted to thecancer cells. These two compounds have activity similar to freeAuristatin F. However, when the antibody is present in Compounds 16 and17, the inhibition of HER2 increases significantly, demonstrating theeffectiveness of the disclosed antibody-drug conjugates in targetingtreatment to specific cells.

Another assay measured the IC₅₀ values for compounds 24 and 25 on thesame cell lines and MCF7 breast cancer cell. Table 2 provides IC₅₀ datafor Compounds 24 and 25 in the above cell lines with Auristatin F,Trastuzumab and Cisplatin as controls. Compound 25 shows a significantincrease in activity as compared to compound 24 which does not comprisea targeting moiety and each of the controls.

TABLE 2 IC₅₀ (nM) Auristatin Cmpd Cmpd Cis- Cell Lines F Trastuzumab 2425 platin HCC1954 200.3 127.7 45.9 0.2 6234.9 NCI-N87 386.3 69.5 91.50.3 1662.2 SK-BR-3 232.6 122.3 56.6 0.1 870.7 BT-474 1543.3 266.8 58.00.4 38517 MCF7 1952.0 447.4 229.7 3.3 9151.0

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the disclosure has referenced specific embodiments, it is apparentthat other embodiments and variations may be devised by others skilledin the art without departing from the true spirit and scope of theinvention. The appended claims are intended to be construed to includeall such embodiments and equivalent variations.

The invention claimed is:
 1. A compound of Formula (I) comprising blockrepeat block monomer (b):

wherein L₁ is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof; L₂ is absent, or canbe of the formula:

L_(2A) is a linking group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, —C(O)—,—NR_(c)—, and any combination thereof; L_(2B) and Lac are independentlyabsent or a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, —C(O)—, —NR_(c)—, and any combination thereof;B_(2A) and B_(2B) are independently absent or a cleavable linker; T is atherapeutic agent selected from the group consisting of chemotherapeuticagents, microtubule inhibitors, DNA-damaging agents and RNAtranscription inhibitors.
 2. The compound of claim 1, comprising from1-372 monomeric units of monomer (b).
 3. The compound of claim 1,wherein the therapeutic agent, T, is one selected from the groupconsisting of auristatin, maytansinoid, taxol, alkaloid, calicheamicin,duocarmycin, doxorubicin, a CC-1065 analog, a methotrexate, apyrrolobenzodiazepine (PBD), tublysin, kinase inhibitor, MEK inhibitor,KSP inhibitor, α-amanitin, β-amanitin, γ-amanatin, and ε-amanatin. 4.The compound of claim 1, wherein the therapeutic agent is an auristatinderivative of structure:

wherein L_(T) is a linking moiety selected from —(CH₂)_(m)—,—(OCH₂)_(m)—, —(CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, and —(CH₂CH₂O)_(m)—, “m” isan integer from 0 to 6; and R_(f) is selected from hydrogen, —NH₂,—C(O)—NH₂, —[C(R_(c))(R_(d))]_(p)—NH₂, —C(O)— [C(R_(c))(R_(d)]_(p)—NH₂,

and “p” is an integer from 1-4.
 5. The compound of claim 3, wherein saidtherapeutic agent is a quinoline alkaloid selected from camptothecin. 6.The compound of claim 1, wherein said therapeutic agent is acamptothecin derivative having the structure:

wherein L_(T) is a linking moiety selected from —(CH₂)_(m)—,—(OCH₂)_(m)—, —(CH₂O)_(m)—, —(OCH₂CH₂)_(m)—, and —(CH₂CH₂O)_(m)—; where“m” is an integer from 0 (i.e. L_(T) is a bond) to 6; and R_(f) isselected from hydrogen, —NH₂, —[C(R_(c))(R_(d))]_(p)—NH₂,

where “p” is an integer from 1-4.
 7. The compound of claim 1, whereinmonomer unit (b) has the structure:


8. The compound of claim 1, wherein the compound has a molecular weightfrom about 10 kDa to about 250 kDa.
 9. A compound comprising blockrepeat block monomer (d):

wherein L₃ is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof; R_(e) is a substituentselected from hydrogen, alkyl and heteroalkyl; L₄ is a group of theformula:

L_(4A) is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, —C(O)—,—NR_(c)—, and any combination thereof; L_(4B) and L_(4C) areindependently absent or a linker group selected from alkylene,heteroalkylene, cycloalkylene, heterocyclylene, arylene, heteroarylene,amidoalkylene, amidoheteroalkylene, —C(O)—, —NR_(c)—, and anycombination thereof; B_(4A) and B_(4B) are independently absent or acleavable linker; C_(4A) is a group selected from

where A is —H or a targeting moiety selected from the group consistingof an antibody, a synthetically functionalized antibody, a peptide and atargeting ligand; “n” is independently at each occurrence an integerranging from 0-5; each cleavable linker B_(2A), B_(2B), B_(4A) andB_(4B), if present, is independently selected from —S—S—, —C(═O)O—,—OC(═O)—, —C(═O)NR_(c)—, —N(R_(c))C(═O)—, —OC(═O)O—, —NR_(c)C(═O)O—,—OC(═O)N(R_(c))— or —N(R_(c))C(═O)N(R_(d))—, —C(═O)N(R_(c))C(═O)—,—C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —OC(═S)O—,—SC(═S)S—, —N(R_(c))SO₂—, —SO₂N(R_(c))—, —N(R_(c))SO₂N(R_(d))—,—C(═O)N(R_(c))N(R_(d))—, —N(R_(c))N(R_(d))C(═O)—,—N(R_(c))N(R_(d))C(═O)O—, —OC(═O)N(R_(c))N(R_(d))—,—C(R_(c))═N—NH—C(═O)—, —C(═O)NH—N═C(R_(c))—, —C(R_(c))═N—O—, —O—

R_(c) and R_(d) are independently selected at each occurrence fromhydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocyclyl.
 10. Thecompound according to claim 9, comprising from 1-186 monomeric units ofmonomer (d).
 11. The compound of 9, wherein the compound comprises oneor more targeting moieties A and A, is an antibody or a syntheticallyfunctionalized antibody specific for an antigen over expressed in cancercells.
 12. The compound of claim 11, wherein the targeting moiety, A, isan antibody or synthetically functionalized antibody specific for anantigen selected from the group consisting of HER-2, EGFR, GPNMB, CD56,TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylylcyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, nectin 4, 5T4,SLTRK6, SC-16, LIV-1, P-Cadherin, PSMA, Fibronectin Extra-domain B,Endothelin receptor ETB, Tenascin c, Collagen IV, VEGFR2, Periostin,CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, CD19 and CD98.
 13. Thecompound of claim 11, wherein the targeting moiety A is trastuzumab or asynthetically functionalized trastuzumab.
 14. The compound of claim 9,wherein C_(4A) is


15. The compound of claim 9, wherein the compound has a molecular weightfrom about 10 kDa to about 250 kDa.
 16. A compound comprising blockrepeat monomer unit (e):

wherein L₃ is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, and any combination thereof; L₄ is a group of theformula:

L_(4A) is a linker group selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, and anycombination thereof; L_(4B) and L_(4C) are independently absent or alinker group selected from selected from alkylene, heteroalkylene,cycloalkylene, heterocyclylene, arylene, heteroarylene, amidoalkylene,amidoheteroalkylene, —C(O)—, —N(R_(c))—, and any combination thereof;B_(4A) and B_(4B) are independently absent or a cleavable linker C_(4A)is a group selected from

A is —H or a targeting moiety selected from the group consisting of anantibody, a synthetically functionalized antibody, a peptide and atargeting ligand; “n” is independently at each occurrence an integerranging from 0-5; R_(c) and R_(d) are independently selected at eachoccurrence from hydrogen, alkyl, heteroalkyl, cycloalkyl, andheterocyclyl; each cleavable linker B_(2A), B_(2B), B_(4A) and B_(4B),if present, is independently selected from —S—S—, —C(═O)O—, —OC(═O)—,—C(═O)NR_(c)—, —N(R_(c))C(═O)—, —OC(═O)O—, —NR_(c)C(═O)O—,—OC(═O)N(R_(c))— or —N(R_(c))C(═O)N(R_(d))—, —C(═O)N(R_(c))C(═O)—,—C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —OC(═S)O—,—SC(═S)S—, —N(R_(c))SO₂—, —SO₂N(R_(c))—, —N(R_(c))SO₂N(R_(d))—,—C(═O)N(R_(c))N(R_(d))—, —N(R_(c))N(R_(d))C(═O)—,—N(R_(c))N(R_(d))C(═O)O—, —OC(═O)N(R_(c))N(R_(d))—,—C(R_(c))═N—NH—C(═O)—, —C(═O)NH—N═C(R_(c))—, —C(R_(c))═N—O—, —O—


17. The compound of claim 16 comprising from 1-186 monomeric units ofmonomer (e).
 18. A pharmaceutical composition comprising at least onecompound of claim 1, or a pharmaceutically acceptable salt or solvatethereof.
 19. A method of inhibiting cancer cells, the method comprisingcontacting the cancer cells with an anti-cancer effective amount of thepharmaceutical composition of claim
 18. 20. A method of treating orinhibiting cancer in a subject, the method comprising administering to asubject in need thereof an anti-cancer effective amount of thepharmaceutical composition of claim 18.